RNA silencing is a remarkable type of gene regulation based on sequence-specific targeting and degradation of RNA. RNA silencing was first discovered in transgenic plants, where it was termed cosuppression or posttranscriptional gene silencing (PTGS). Only recently a sequence-specific RNA degradation process, RNA interference (RNAi), related to PTGS has been found in ciliates, fungi and a variety of animals from C. elegans to mice and human cells. Although they may differ in detail, RNAi and PTGS result from the same highly conserved mechanism, indicating an ancient origin. The basic process involves a double stranded RNA (dsRNA) that is cleaved into small double stranded interfering RNAs (siRNA) which guide recognition and targeted cleavage of homologous mRNA. These small dsRNAs resemble breakdown products of an RNase III-like digestion. In particular, siRNAs are target-specific short double stranded RNAs wherein each strand of the siRNAs carries 5′monophosphate, 3′hydroxyl termini and 3′ overhangs of 2-3 nucleotides (Caplen, N. et al., 2001, PNAS (98) 9742-9747).
RNAi has attracted considerable attention because it is a means of knocking out the activity of specific genes, being particularly useful in species that were previously considered not to be amendable to genetic analysis. Recent studies demonstrated that synthetic siRNAs can induce gene-specific inhibition of expression in C. elegans and in cell lines from humans and mice (Caplen N., et al., 2001, PNAS (98) 9742-9747; Elbashir S., et al., 2001, Nature (411) 494-498). In said publications it was further demonstrated that in mammalian cells siRNAs provide a sequence specific answer compared to the use of longer dsRNAs which inactivate the translation factor eIF2α, leading to a generalized suppression of protein synthesis. Also, in comparison to inhibition of gene expression using antisense technology, siRNAs seem to be very stable and thus may not require the extensive chemical modifications that single stranded RNA antisense oligonucleotides require to enhance the in vivo half life.
It is therefore to be expected that RNA silencing using siRNAs will become an important tool in engineering control of gene expression as well as in functional genomics and a variety of biotechnology applications ranging from molecular farming to possibly even gene therapy in animals. As different siRNAs may work with different effectiveness on their targets, the testing of more than one siRNA for a particular target will be desirable. In addition, genome-scale reverse genetics programs will require large numbers of siRNAs.
However, production of double stranded target-specific RNA oligos by traditional chemical synthesis remains relatively slow and expensive when compared to DNA oligo synthesis. In addition, chemical synthesis of RNA oligos requires special synthesizers and complex purification protocols. The present invention provides an alternative approach to produce short double stranded target-specific RNAs based on in vitro transcription using bacteriophage or other viral polymerases and target sequence-specific oligonucleotide templates. Compared to the chemical synthesis of RNA oligos the present invention is relatively quick and easy to perform.
However, the in vitro transcribed siRNAs differ from the chemically synthesized RNA oligos in two ways. Primarily, identical to the natural occurring siRNAs, the chemically synthesized RNA oligos have a 5′monophosphate group. The in vitro transcribed siRNAs retain a 5′triphosphate group. It was unknown whether the presence of this triphosphate group renders the in vitro transcribed siRNAs incompetent to induce RNA interference.
Secondly, chemically synthesized RNA oligos are highly purified using amongst others Ion Exchange and Reverse Phase HPLC wherein purity and quality of the synthesized compounds is further evaluated using amongst others NMR and mass spectrometry analysis. In the present invention a simple, crude purification protocol is used comprising size exclusion chromatography, phenol:chloroform extraction and ethanol precipitation. It was again uncertain whether the ommitance of an extensive purification protocol would affect the usefulness of “in vitro” transcribed RNAs in RNA-mediated silencing.
Surprisingly, the present invention demonstrates that the 5′triphosphate group and the crude purification does not affect the RNA silencing activity of “in vitro” transcribed RNAs and provides an alternative approach to siRNA synthesis which makes it accessible as a research tool in an average molecular biology laboratory.
Existing in vitro methods to synthesize small single stranded RNAs of defined length and sequence (Milligan F. et al., 1987, Nucleic Acid Res. (15) 8783-8798), were not directly applicable for the synthesis of small interfering RNAs. The problem resides in the fact that RNA polymerases tend to transcribe some nucleotides from the promoter sequence into the transcript. As a consequence, the target-specific dsRNAs which can be produced by annealing complementary single stranded RNA molecules generated using the aforementioned methods, must comprise at the 5′end the nucleotides transcribed from the promoter sequence and at the 3′end the nucleotides complementary to the nucleotides transcribed from the promoter sequence. It may well be that in the mRNA of the target sequence no stretch of a defined sequence length exists wherein the 5′-end consists of the nucleotides transcribed from the promoter sequence and the 3′-end of the nucleotides complementary to the nucleotides transcribed from the promoter sequence. The present invention solves this problem by providing truncated RNA polymerase promoter sequences wherein one or more nucleotides at the 5′end of the template strand of the promoter sequence are replaced by nucleotides that are part of the target-specific sequence. These substitutions do not affect the in vitro transcription yields, but increase the possibility that at least one target-specific sequence of a defined sequence length exists in the mRNA of the target protein, wherein the 5′-end consists of the nucleotides transcribed from the promoter sequence and the 3′-end of the nucleotides complementary to the nucleotides transcribed from the promoter sequence.
This and other aspects of the invention will be described herein below.